Nearly all nucleic acid detection systems rely upon the use of nucleic acid hybridization probes. The applications of nucleic acid hybridization probes are well recognized in numerous fields, including medical diagnostics, molecular medicine, forensic science, specimen and organism cataloging and microbial pathogen epidemiology. The challenges in designing nucleic acid hybridization probes have remained the same in nearly all applications, namely achieving probe designs having high affinity and specificity for a given target and displaying high signal specific activity for identifying the target.
Whole genome screening methodologies using nucleic acid hybridization probes illustrate these challenges. In particular, chromosomal screening to identify nucleic acid targets in both a sequence-specific and chromosome-specific manner poses the greatest challenge for nucleic acid hybridization probe design. One such method, fluorescence in situ hybridization (FISH), uses nucleic acid hybridization probes having both superior targeting specificity and high signal specific activity to visualize the location of nucleic acid targets within a chromosomal spread. Thus, probes that may otherwise be suitable for other hybridization applications may not be necessarily appropriate for FISH applications.
Methods of preparing nucleic acid hybridization probes for FISH applications are well known in the art. For short probes prepared through chemical synthesis (e.g., single copy probes having a length less than 200 nucleotides), the probes are synthesized that contain labels or moieties that may be subsequently reacted with labels. Alternatively, such probes have been post-labeled following their synthesis using any suitable enzymatic or chemical means.
Long probes, such as those having a length from 200 bp to 500 kbp have been prepared in numerous ways. Nucleic acid substrates for long probes have been fragmented using sonication or restriction enzyme digestion. The resultant fragment population can be chemically modified to incorporate a suitable label.
Alternatively, the fragment population can be enzymatically labeled with nucleotide triphosphate analogs that contain a label or a moiety for reacting with a label. Suitable enzymatic labeling procedures have included internal labeling methods or terminal labeling methods. Known examples of internal labeling methods include polymerase-mediated techniques, such as nick-translation protocols, random priming methods, and the polymerase chain reaction. Known terminal labeling methods include polynucleotide kinase methods, ligase techniques and terminal transferase procedures.
Nucleic acid hybridization probes are typically composed of DNA or DNA analogs, such as PNA. RNA has also served as nucleic acid hybridization probes, owing to the fact that RNA transcripts can be produced in high yield as single-stranded molecules. Yet RNA-based probes often suffer in quality and reproducibility, owing to their sensitivity to nucleolytic degradation in biological specimens used in FISH assays.
Nucleic acids used for long probes are usually maintained in cloning vectors. For example, vector-borne nucleic acids are typically propagated in bacterial cultures. Typical harvests of such nucleic acids are relatively inefficient from such cultures. Thus, considerable time and expense is required to maintain a source of material for long probes.
Furthermore, prior art nucleic acid hybridization probes for FISH applications usually lack robust signal generating capability. This is attributed in part to the inherently low specific activity of label incorporation into a probe in relation to the suitability of the probe for hybridization in FISH assays. A high number of labels incorporated into a given nucleic acid can result in signal reduction due to internal quenching as well as lower hybridization specificity of the resultant probe. Moreover, a very long probe is often needed to place sufficient signal on a target nucleic acid. Yet as the length of such probes increase (holding probe mass constant), the concentration of each individual segment decreases. Thus, longer probes require longer incubation times for each hybridization segment to hybridize to its complement sequence within the nucleic acid target.
Thus, there is a need in the art for improved nucleic acid hybridization probes, both from the standpoint of improved production yields and probe designs having superior signal generating capability.